Phosphatidylinositol polyphosphates (PPIs) have recently emerged as key regulators of membrane trafficking pathways. PPIs transiently appear at specific membranes at specific times. However, how their transient appearance is regulated, and the roles that these molecules play in membrane traffic, cytoskeletal rearrangements, ion homeostasis and other events are not well understood. Phosphatidylinositol (3,5)- bisphosphate (PI3,5P2) is found in all eukaryotes, from yeast to humans. Depletion of PI3,5P2 leads to perinatal lethality and massive neurodegeneration in mice. Large vacuoles form in the cell bodies of neurons in both the central and peripheral nervous system. Yeast lacking PI3,5P2, also form large vacuoles and have additional defects in vacuole inheritance, retrograde traffic from the vacuole, and in ion homeostasis. PI3,5P2 is generated by PI3P by the kinase Fablp. In yeast, PI3,5P2 levels are 18-28 fold lower that the other PPIs. However, within minutes of exposure to hyperosmotic stress, PI3,5P2 levels rise more than 20-fold and then by 30 min return to basal levels. Specific stimuli induce stress profound elevation of PI3,5P2 in animal cells as well. In yeast, osmotic stress signals though a large protein complex containing Vac14, Fig4, Vac7 and Atg18, and results in the transient activation of the PI3P 5-kinase Fab1. With the exception of Vac7, all of the above proteins have direct human homologues. Thus, there is a common capacity and mechanism for the control of PI3,5P2 levels. Fab1 and Vac14 are expressed in all tissues, thus it is likely that PI3,5P2 in humans may regulate both normal endosomal function and also specialized membrane trafficking pathways. We will use the distinct advantages afforded by yeast and mammalian cells to elucidate the regulation and roles of PI3,5P2. The goals of this grant are to 1) determine how levels of PI3,5P2 are regulated, 2) determine the down-stream pathways that are regulated by PI3,5P2, and 3) initiate studies to determine why loss of PI3,5P2 causes neurodegeneration in mice.